Editing
Dinh/Dinh 2015/NOTES/2015-9-15
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
= 1 ng RRBS protocol from plasma DNA = * Combined plasma DNA yielded 30 uL x 1.64 ng/uL * Test different input: 2 ng, 1 ng, 0.5 ng, NTC. Perform duplicates of each. * Mostly following Noi's protocol (Noiβs 1 ng input protocol from LabWiki: 2014-6-29). ===MspI digestion=== <nowiki>MspI (10U/uL) 1.00 uL 10x Tango Buffer 2.00 uL 1.2pg/ul unmeth lambda DNA 0.83 uL (should be 12pg/uL but I used the wrong one) 1 ng DNA input 6.00 uL (varying 0.5ng-2ng) Nuclease free water 8.17 uL</nowiki> *Spin down the tubes, mix by gentle pulse vortexing on PCR 10x *Spin down the tubes, *Incubate 37C O/N (~13 hr) β 65C 20 min β hold at 4C ===End-repair/dA-tailing=== <nowiki>MspI digested DNA 18.00 uL Klenow exo- 1.00 uL dATP:dCTP:dGTP (20mM,2mM,2mM) 1.00 uL</nowiki> *Spin down the tubes, mix by gentle pulse vortexing on PCR rack 10x *Spin down the tubes, *Incubate 30C 20 min β 37C 20 min β hold at 4C (no heated lid) *Heat inactive 75C 10 min β 4C (with heated lid) ===Methylated adaptor ligation=== <nowiki>dA-tailed DNA 20.00 uL 10X Tango Buffer 0.50 uL HC T4 DNA ligase(30U/uL) 1.00 uL 10mM ATP 1.25 uL Nuclease free water 1.25 uL 1:20 diluted TruSeq adaptors 1.00 uL</nowiki> *TruSeq barcodes <nowiki> NTC_1 18 NTC_2 19 2ng_1 20 2ng_2 21 1ng_1 22 1ng_2 23 0.5ng_1 25 0.5ng_2 27</nowiki> *Spin down the tubes, mix by gently pulse vortexing on PCR rack 10x *Spin down the tubes, *Incubate 16C, 22 h (no heated lid, >=20 hr) *Mix by gentle pulse vortexing on PCR rack 5x *Spin down *Heat inactivate at 65C, 20 min β hold 4C ===AMPure beads purification=== * Need to confirm with Noi whether she used beads to purify the adaptor ligated DNA. * Add 5 uL of water to make total volume equals 30 uL of ligation mixture. * Add 60 uL of AMPure beads and repipette 10X to mix. * Incubate for 30 minutes at room temperature. * Wash twice with freshly prepared 70% ethanol. * Dry for 15 minutes at room temperature. (While drying, label and UV new tubes for bisulfite conversion below). * Add 30 uL of EB buffer to elute. ===Bisulfite conversion using LifeTech MethylCode Bisulfite conversion kit=== * Prepare 3 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer. **If CT Conversion reagent prepare with 800ul H2O, need to add 5ul of H2O to 25ul adaptor-ligated DNA and added 120ul of CT conversion reagent. * Mix well and spin down the tube * Put on thermomixer at 37C for 15min, 1,000rpm * Solution should be clear * Spin down the tube * Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul * Mix by pipetting 10X with multi-channel pipette * Spin down the tube * Incubate following below program 98Β°C for 10 minutes (DNA denaturation) 64Β°C for 2.5 hours (Bisulfite conversion) 4Β°C storage for up to 20 hours or continue to desulfonation * Add 600ul Binding Buffer to each column * Adjust p200 to 200ul and transfer bisulfite treated DNA to the column * Mix by pipetting 5x * Spin down column at 14,000 rpm for 30s * Discard spnt * Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube) * Spin down column at 14,000 rpm for 30s (do not discard spnt) * Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube). Incubate for 15-20 minutes at room temperature. * Spin down column at 14,000 rpm for 30s (do not discard spnt) * Add 200ul Wash Buffer * Spin down column at 14,000 rpm for 30s * Discard spnt * Add 200ul Wash Buffer * Spin down column at 14,000 rpm for 3min (do not discard spnt) * Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube * Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min) * Wait for 2min * Spin down column at 12,000 rpm for 1min * Continue to amplification (During elution step, PCR mix should be prepared) ===Amplification === * Perform amplification with and without Sybr. I reduced reaction volumes by half each. * Perform PCR with 12 cycles (fixed) <nowiki> bis-cvt DNA 15.000 uL 10X Reaction buffer 2.500 uL dNTP, 10mM 0.625 uL TruSeq F/R, 10uM 0.750 uL 50X Sybr 0.400 uL (0 for no sybr) PfuTurboCX 0.500 uL Nuclease free water 5.225 uL (5.625 for no sybr) </nowiki> *Mix well *Program: 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min *Purify with 1X AMPure beads, incubate DNA with beads 15 minutes, wash twice with freshly prepared 80% ethanol, dry beads for 20 minutes (?)) *Elute with 20 uL EB buffer *Continue to 2nd round PCR. No PAGE Verification * Test 2nd round PCR * Noi's protocol: <nowiki> Purified 1st round DNA 3.00 uL 5X Phusion HF buffer 2.00 uL dNTP mix, 10mM 0.25 uL TruSeq F/R, 10 uM 0.30 uL 50X Sybr 0.08 uL (0 for no sybr) Phusion HF 0.10 uL Nuclease free water 4.27 uL (4.35 for no sybr) </nowiki> * I couldn't find Phusion HF, so I used Phusion Hotstart Flex Master Mix instead. *Mix well *Program: 98C for 2min β [98C for 10sec β 60C for 30sec β 72C for 1min] X 9X cycles β 72C for 2min *Load everything to 6% gel and run at 225 V for 30 min. * Once PCR cycle is determined (8-9 cycles?), prepare PCR for all the samples and then purify with AMPure beads. Run gel to also determine the pooling ratios and perform size selection for 150-400 bp bands. * Gel images: ** (+) is with sybr added, (-) is without sybr added. The amplification patterns are similar on gel, except in some cases sybr added more intensity. ** I remember that PfuTurboCx have less amplification with sybr, its not possible that sybr would increase the amplification efficiency, so I think the sybr green may have helped to make the fluorescence brighter for the DNA amplified with sybr. For now, I don't see evidencee that the sybr have an effect on amplification, so I could amplify with or without sybr. [[File:Dinh_1ng_RRBS_09182015_gel1.jpg | 300px]] [[File:Dinh_1ng_RRBS_09182015_gel2.jpg | 300px]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information